LaMYC7, a positive regulator of linalool and caryophyllene biosynthesis, confers plant resistance to Pseudomonas syringae

Abstract Linalool and caryophyllene are the main monoterpene and sesquiterpene compounds in lavender; however, the genes regulating their biosynthesis still remain many unknowns. Here, we identified LaMYC7, a positive regulator of linalool and caryophyllene biosynthesis, confers plant resistance to Pseudomonas syringae. LaMYC7 was highly expressed in glandular trichomes, and LaMYC7 overexpression could significantly increase the linalool and caryophyllene contents and reduce susceptibility to P. syringae in Nicotiana. In addition, the linalool possessed antimicrobial activity against P. syringae growth and acted dose-dependently. Further analysis demonstrated that LaMYC7 directly bound to the promoter region of LaTPS76, which encodes the terpene synthase (TPS) for caryophyllene biosynthesis, and that LaTPS76 was highly expressed in glandular trichomes. Notably, the LaMYC7 promoter contained hormone and stress-responsive regulatory elements and responded to various treatments, including ultraviolet, low temperature, salt, drought, methyl jasmonate, and P. syringae infection treatments. Under these treatments, the changes in the linalool and caryophyllene contents were similar to those in LaMYC7 transcript abundance. Based on the results, LaMYC7 could respond to P. syringae infection in addition to being involved in linalool and caryophyllene biosynthesis. Thus, the MYC transcription factor gene LaMYC7 can be used in the breeding of high-yielding linalool and caryophyllene lavender varieties with pathogen resistance.


Introduction
During plants' lives, they experience various environmental pressures, including biotic stressors (e.g.pathogens) and abiotic stressors (e.g.cold) [1,2].Plants have developed various defense systems against these stressors in order to survive and produce the next generation [3].Among the several biotic stressors, pathogens pose the greatest risk to plant growth, development, and yield.Pseudomonas syringae, a gram-negative pathogenic bacterium, affects plants worldwide [4,5].Volatile terpenoids mount an effective defense in response to multiple stresses [6].Volatile terpenoids, including monoterpenoids (e.g.linalool, pinene, myrcene, and linalyl acetate) and sesquiterpenoids (e.g.caryophyllene, farnesene, and germacrene), are the most common classes of volatile plant terpenoids.A high prevalence of linalool and caryophyllene is found in the plant kingdom, in general, and the Lamiaceae family, in particular [7,8].
Linalool (C 10 H 18 O), an acyclic monoterpenoid, has ecological functions, such as serving as an attractant for both pollinators [9,10] and predators [11], and acts as an antiherbivore defense to protect plants from damage [12,13].The compound is widely used in the pharmaceutical, cosmetic, food, and cleaning-product industries because of its pleasant scent, antibacterial properties, and sedative effects, among other properties [14][15][16][17][18][19].Caryophyllene (C 15 H 24 ; β-caryophyllene) is a bicyclic sesquiterpenoid with seem-ingly innumerable biological properties and commercial applications.In plants, β-caryophyllene contributes to lateral root formation, increases pathogen resistance, and provides plant resistance by jasmonic acid (JA) [20][21][22].It is used as a fragrance or f lavor compound in the cosmetics and food industries.Pharmacological studies have shown that β-caryophyllene has local anesthetic and anti-inf lammatory effects, is used to treat depression and general anxiety, and can repel insects as well [23,24].
The terpenoid biosynthetic pathway in plants has received a great deal of attention.Typically, geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) are synthesized by the 2-C-methyl-derythritol 4-phosphate (MEP) pathway in plastids and the mevalonate pathway (MVA) in the cytoplasm, respectively.Different terpene synthases (TPSs) convert these compounds into monoterpenoids or sesquiterpenoids [25][26][27].However, studies on the transcriptional regulation of volatile terpenoids are fewer than those on volatile terpenoid biosynthesis.
Transcription factors (TFs) control transcription or the simultaneous expression of several genes by binding to certain DNA sequences.Thus, TFs are considered the best targets for pathway engineering [28].MYC genes play a pivotal role in secondary metabolite accumulation and are critical transcriptional activators that respond to JA signaling [29].In Arabidopsis, AtMYC2 controls the transcript abundance of AtTPS11 and AtTPS21 to regulate caryophyllene biosynthesis [30,31], and in the presence of JA, caryophyllene also equips plants with the ability to resist P. syringae pv.tomato (Pst) DC3000 [22].However, the transcriptional regulation of volatile terpenoid remains elusive [32,33].The model plant, Arabidopsis thaliana has no glandular trichomes (GTs), which produce and accumulate terpenoids, and its terpenoid species are few.In contrast, lavender (Lavandula angustifolia), with more than 75 volatile terpenoids [34,35], can serve as a model plant to study terpenoid regulation because a chromosome-based 'Jingxun 2' lavender genome has already been published [36].
In this study, by using RNA-sequencing, transgenic technology, solid-phase micro extraction coupled with gas chromatographymass spectrometry (SPME-GC-MS), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme activity, yeast one-hybrid (Y1H), and dual-luciferase (dual-LUC) assays, we comprehensively analyzed the expression of LaMYC7 and its regulatory role in terpenoid synthesis in order to reveal its functions in protecting plants to cope with adversity, especially against P. syringae infection.Our findings not only serve as a basis for understanding how volatile terpenoid biosynthesis is controlled in lavender but also open the door to deciphering the transcriptional regulation of volatile terpenoids, as well as suggest that LaMYC7 is a candidate gene for developing high-yielding and pathogenresistant lavender plants.

LaMYC7 isolation and bioinformatics analysis
Based on L. angustifolia genomic data (PRJNA642976), twentysix MYCs were obtained using the hidden Markov model with PF14215 and PF00010 as queries (Supplementary Data Table S1), and the transcript abundance of the MYC gene LaMYC7 increased after methyl jasmonate (MeJA) treatment (Fig. 1A).Compared with other tissues, the transcript abundance of LaMYC7 was noticeably higher in leaf and GT, and gene expression was higher during FB0 and F4 f lower development (Fig. 1B, C).The LaMYC7 coding DNA sequence (CDS) was 1965 bp, encoding 654 amino acids (aas) (Fig. 1D, E).According to our bioinformatics study, LaMYC7 had a basic helix-loop-helix (bHLH)-MYC sequence (78-258 aa) and DNA-binding domain (475-552 aa) (Fig. 1E).The physicochemical properties of LaMYC7 were analyzed using ExPASy, and the isoelectric point and molecular weight of the protein were 5.31 and 71.25 kDa, respectively.According to the AtbHLH classification, LaMYC7 was clearly in the subfamily 2 or subgroup III (d + e) (Fig. 1D).

Subcellular localization and transactivation activity of LaMYC7
Subcellular localization of LaMYC7 in Nicotiana (Nicotiana benthamiana) leaves was determined by a transient expression test.35S:LaMYC7-GFP was found exclusively in the nucleus of plant cells, while the empty vector (35S::GFP) was located in the nucleus and cytoplasm (Fig. 2A), indicating that LaMYC7 is localized in the nucleus.
The transactivation activity of LaMYC7 was evaluated using AH109 yeast cells and the pGBKT7 vector.AH109 cells transformed with each vector were grown on SD/−Trp medium.AH109 cells with the negative control vector (pGBKT7) did not appear blue, whereas AH109 cells with the recombinant pGBKT7-LaMYC7 vector or positive control vector (pGBKT7-p53) turned blue on SD/−Trp/X-α-Gal medium (Fig. 2B), indicating that LaMYC7 has transactivation activity.

LaMYC7 overexpression in Nicotiana increases volatile terpenoid biosynthesis
To assess the role of LaMYC7 in volatile terpenoid biosynthesis, LaMYC7 was overexpressed in Nicotiana and the T2 generation of transgenic lines #2 and #9 was selected for further research.Terpenoid content and gene expression levels were measured using SPME-GC-MS and qRT-PCR, respectively.The total volatile terpenoid, sesquiterpenoid, and monoterpenoid contents were significantly increased, which was in accordance with LaMYC7 overexpression in Arabidopsis (Supplementary Data Fig.S1, 2).Notably, the linalool and caryophyllene levels were approximately 0.71-and 1.98-fold higher in LaMYC7-overexpressing lines #2 and #9, respectively, compared with control 2300 plants (Fig. 3A-F and Supplementary Data Fig.S3).In addition, the relative expression levels of NtHMGL and NtFPPS, key enzymes that control sesquiterpene biosynthesis, were decreased in the f lowers of LaMYC7-overexpressing Nicotiana lines #2 and #9 compared with control 2300 plants (Fig. 3G, H).However, the relative expression levels of NtDXS, NtDXR, and NtGPPS, key enzymes that control monoterpene biosynthesis, were significantly increased in LaMYC7-overexpressing Nicotiana lines #2 and #9 compared with control 2300 plants (Fig. 3I-K).Moreover, the expression levels of linalool synthase (NtTPS67) and caryophyllene synthase (NtTPS7) were consistent with linalool and caryophyllene accumulation, which was significantly increased in LaMYC7-overexpressing Nicotiana lines #2 and #9 compared with control plants (Fig. 3L, M).However, LaMYC7 overexpression in Arabidopsis could increase the linalool content but not significantly, while the caryophyllene content significantly increased (>7-fold) (Supplementary Data Fig.S4A-F and S5).The relative gene expression levels of key enzymes that control terpenoid biosynthesis and some TPSs showed that LaMYC7 overexpression in Arabidopsis could significantly increase the expression levels of AtHMGR1, AtFPPS1, AtDXR, AtGPPS, and caryophyllene synthase (AtTPS21) (Supplementary Data Fig.S4G-M).
In transgenic Nicotiana, the zeatin riboside (ZR), indole acetic acid (IAA), and JA levels were significantly decreased compared with control plants.However, the changes in the gibberellin (GA 3 ) content were not significant, and the abscisic acid (ABA) content significantly increased (Supplementary Data Fig.S6).LaMYC7 overexpression had no effect on plant height or total anthocyanin content (TAC) in transgenic Nicotiana, but chlorophyll and carotenoid biosynthesis decreased (Supplementary Data Fig.S7).

LaMYC7 overexpression confers Nicotiana with resistance to P. Syringae
The phenotypes of LaMYC7-overexpressing lines (i.e.#2 and #9) and control plants (i.e.wild-type (WT) and 2300) inoculated with Pst DC3000 were studied after 5 days to elucidate the potential biological role of LaMYC7 in plant disease prevention.Necrotic spots were found in WT and empty vector plants, while LaMYC7overexpressing lines grew normally (Fig. 4A).In addition, bacterial growth on plants was assessed using Pst DC3000.The outcomes of the statistical analysis revealed that the bacterial population was dramatically decreased in LaMYC7-overexpressing lines compared with control plants (Fig. 4B, C).Furthermore, the antimicrobial activity of linalool and caryophyllene against Pst DC3000 growth was evaluated.Linalool inhibited Pst DC3000 growth in a dose-dependent manner and showed strong antimicrobial activity regardless of Pst DC3000, while caryophyllene did not show any antibacterial activity regardless of Pst DC3000 at a concentration of 40 μl/ml (Fig. 4D, E).

Isolation and characteristics of caryophyllene synthase
Based on genomic data (PRJNA642976), 100 TPSs were previously found in L. angustifolia [34].The TPS genes LaTPS26 (La05G01453) and LaTPS76 (La22G02785) were present in the turquoise module along with LaMYC7 in a weighted correlation network analysis (WGCNA) (unpublished).In the gene expression profiling of various tissues, LaTPS26 and LaTPS76 had the highest levels of expression in GTs, and the expression level of LaTPS76 was significantly greater than that of LaTPS26 (Supplementary Data Fig.S8).The open reading frames of LaTPS26 and LaTPS76 encoded 549-and 540-aa proteins, respectively.The protein sequences of LaTPS26 and LaTPS76 contained DDxxD and (N, D) D (L, I, V) x (S, T) xxx E motifs, which are the typical TPS domains (Fig. 5A).Protein subcellular localization prediction (WoLF PSORT; https://wolfpsort.hgc.jp/) showed that LaTPS26 and LaTPS76 were localized in the cytoplasm.To confirm the subcellular localization of LaTPS26 and LaTPS76, LaTPS26-GFP and LaTPS76-GFP fusion proteins were produced and transformed into Agrobacterium tumefaciens GV3101.35S::GFPs were identified in both cytoplasm and nucleus, but LaTPS26-GFP and LaTPS76-GFP were identified exclusively in the cytoplasm (Fig. 5B), showing that LaTPS26 and LaTPS76 localize in the cytoplasm.
To ascertain the biological roles of LaTPS26 and LaTPS76, the enzyme activity of the LaTPS26 protein and LaTPS76overexpressing transgenic Arabidopsis were characterized.Enzyme activity assays demonstrated that LaTPS26 can convert FPP to caryophyllene (Fig. 6A-C).In addition, the caryophyllene content significantly increased in LaTPS76-overexpressing transgenic plants compared with control plants (Fig. 6D-I).In addition, we found four homologous genes of linalool synthase (based on the published 'Jingxun 2' lavender genome) which were highly expressed in GTs.However, among these four genes, the highest FPKM value was 235 (Supplementary Data Fig.S9).

LaMYC7 directly binds to the LaTPS76 promoter
Because LaMYC7, LaTPS27, and LaTPS76 were related to caryophyllene biosynthesis, the regulatory connection between LaMYC7 and LaTPS26/LaTPS76 was examined.The promoters were also inserted into pLacZi vectors (Fig. 7A).After being co-transformed with the pB42AD-MYC7 or empty pB42AD vector, the Y1H assay results showed that all co-transformed cells survived on SD-Trp/Ura medium, while only co-transformed cells with pLacZi-TPS76 and pB42AD-MYC7 turned blue on SD-Trp/Ura/X-gal medium (Fig. 7B).This implied that LaMYC7 could directly bind to the LaTPS76 promoter but was unable to bind to the LaTPS26 promoter.Furthermore, the LaTPS76 promoter was inserted into the dual-LUC reporter plasmid containing firef ly luciferase (FLuc) and Renilla luciferase (RLuc) reporter genes (Fig. 7C).LaMYC7 could also significantly activate the LaTPS76 promoter (Fig. 7D).

Analysis of the LaMYC7 promoter sequence and stress response
The LaMYC7 promoter sequence, a sequence 2000-bp upstream from the LaMYC7 translation initiation site, was evaluated by PlantCARE software (Supplementary Data Table S2).Four ABAresponsive elements were located at +776, −1039, +777, and +1040 bp.One TATC-box, which responds to GA, was found at +1128 bp.One TC-rich repeat element, which participates in stress response and defense, was found at +1455 bp.Two MeJAresponsiveness elements were found at −1666 and +1666 bp (Fig. 8A and Supplementary Data Table S2).Furthermore, LaMYC7 confers plant tolerance to drought stress in LaMYC7overexpressing plants (Supplementary Data Fig.S10).

Discussion
Linalool and caryophyllene have multiple ecological functions, including chemical signals for plant-pollinator interactions, antiherbivory, and pathogen resistance [9][10][11]22].In addition to their ecological functions, these compounds find applications in the pharmaceutical, cosmetic, and food industries [14-16, 23, 24].Linalool and caryophyllene are formed by TPSs using GPP and FPP as substrates [37,38]; however, the transcriptional regulation mechanism underlying their biosynthesis is unclear.In the model plant Arabidopsis, AtMYC2 participated in regulating caryophyllene biosynthesis by binding to TPS21 and TPS11 promoters to regulate their expression [30].LaMYC4 (now called LaMYC17) overexpression increased the caryophyllene content in tobacco [39], whereas the caryophyllene content increased in SlMYC1-downregulated lines, and FhMYC2 interacted with FhMYB21 to regulate the expression of TPS1, which produces linalool [40,41].We extensively searched the lavender genome and identified 26 putative MYC TFs (LaMYC1-26) containing the bHLH-MYC_N and bHLH domains.Gene expression analysis indicated that LaMYC7 was differentially expressed after MeJA treatment (upregulation) and highly expressed in GTs (Fig. 1A, B).Therefore, LaMYC7 was further analyzed.
LaMYC7 overexpression resulted in increased linalool and caryophyllene contents in Nicotiana (Fig. 3A-F and Supplementary Data Fig.S4A-F).In addition, the transcript levels of NtTPS67 and NtTPS7, the structural genes of the linalool and caryophyllene biosynthetic pathway, were significantly increased in LaMYC7overexpressing lines (Fig. 3L, M).The transcript levels of the MEP pathway key genes NtDXS and NtGPPS were significantly increased, while the transcript level of NtHMGRL, a key gene of the MVA pathway, was significantly decreased (Fig. 3G-K).These results indicated that LaMYC7 regulated linalool and caryophyllene biosynthesis and inf luenced carbon f low in the MEP pathway.Notably, LaMYC7 overexpression resulted in enhancing the transcript levels of some early pathway genes (e.g.AtHMGR1, AtFPPS1, AtDXS, AtDXR, and AtGPPS) in Arabidopsis (Supplementary Data Fig.S4G-K).The caryophyllene content and caryophyllene synthase transcript level were significantly increased, while the linalool content and linalool synthase transcript level did not change significantly (Supplementary Data Fig.S4A-F, L, M).Volatile terpenoids also need specialized storage structures to prevent autocytotoxicity; for example, monoterpenoids are harmful to unspecialized plant cells and must be sequestered [42,43].Because Arabidopsis lacks GTs, it has fewer volatile compounds, especially monoterpenoids such as linalool.These may be the reasons for the difference in the results of LaMYC7 overexpression in plants with and without GTs.
In addition to the role of LaMYC7 in linalool and caryophyllene biosynthesis, its role in regulating stress was investigated.Plants are subjected to various stresses during their entire lives, and they have thus developed several defense mechanisms to withstand these stresses [44][45][46].The LaMYC7 promoter analysis results suggested that LaMYC7 plays an important role in environmental  adaptation.Furthermore, the results of UV, MeJA, drought, low temperature, salt, and P. syringae infection treatments indicated that LaMYC7 can respond to multiple stresses (Fig. 8A, B).LaMYC7 overexpression in Nicotiana could significantly increase resistance to P. syringae (Fig. 4A-C), which causes economically important plant diseases [4].Caryophyllene has been shown to confer plant resistance to P. syringae infection through the JA signaling pathway [22].However, LaMYC7 overexpression in Nicotiana decreased JA levels (Supplementary Data Fig.S6).The negative feedback mechanism enables plants to achieve a dynamic balance to ensure normal growth.The antibacterial activity results showed strong antimicrobial activity irrespective of the presence of P. syringae at concentrations exceeding 16 μl/ml and dose-dependently; however, caryophyllene did not show any antibacterial activity regardless of Pst DC3000 at a concentration of 40 μl/ml (Fig. 4D, E).Therefore, LaMYC7 overexpression conferred plant resistance to P. syringae, which may have been achieved by linalool.The intricacy of terpenoid regulation is illustrated by the fact that TPSs frequently have numerous copies to assist complex metabolic processes [47].The TPS genes LaTPS26 (La05G01453) and LaTPS76 (La22G02785) in this study were found in the turquoise module with LaMYC7 by WGCNA (unpublished).According to the results of the gene expression profiling of various tissues, LaTPS26 and LaTPS76 had the highest levels of expression in GTs, and the expression level of LaTPS76 was significantly greater than that of LaTPS26 (20-fold) (Supplementary Data Fig.S8).Transgenic and in vitro enzyme activity analysis showed that both LaTPS26 and LaTPS76 can synthesize caryophyllene (Fig. 6).Notably, the results of the Y1H and LUC assays showed that LaMYC7 can only bind to the LaTPS76 promoter, indicating that LaMYC7 regulates caryophyllene synthesis by binding to the LaTPS76 promoter.In addition, the tissue expression analysis results showed that the four homologous genes of linalool synthase were highly expressed in GTs, FPKM of that were far lower than LaTPS76 (Supplementary Data Fig.S9).However, in L. angustifolia essential oil, the content of linalool was more than 20-fold that of caryophyllene [48,49].Thus, the four homologous genes of linalool synthase may not be major-effect genes Fig. S9.Therefore, further research is needed to elucidate linalool regulation by LaMYC7.

Conclusions
To our knowledge, LaMYC7, isolated from the lavender cultivar 'Jingxun 2'in this study, is the first MYC TF shown to be a positive regulator of linalool and caryophyllene biosynthesis as well as a disease resistance-responsive MYC TF that actively regulates disease resistance.In addition, LaMYC7 bound to the promoter of LaTPS76, which produces caryophyllene.Furthermore, LaMYC7 overexpression conferred plant resistance to Pst DC3000, while the JA level decreased.Linalool exhibited strong antibacterial activity against Pst DC3000 growth in vitro.Thus, we identified a novel MYC TF, LaMYC7, which has roles in linalool and caryophyllene biosynthesis as well as disease resistance in lavender.

Subcellular localization and transactivating activity of LaMYC7
The primer pairs (Supplementary Data Table S4) used for LaMYC7, LaTPS26, and LaTPS76 were based on their full-length CDSs obtained from the lavender genome (PRJNA642976) and the KpnI restriction site sequence of the pCAMBIA2300 vector.LaMYC7, LaTPS26, and LaTPS76 were isolated from sepal cDNA using PCR.The PCR products were recombined into the empty vector pCAMBIA2300 to produce the recombinant vectors 35S::LaMYC7-GFP, 35S::LaTPS26-GFP, and 35S::LaTPS76-GFP.A. tumefaciens GV3101 was heat-shock transformed with the empty and vectors for subcellular localization analysis.As previously described [51], N. benthamiana leaves were also transformed with the recombinant vectors and the empty vector 35S::GFP.After 3 days, subcellular localization in the leaves was analyzed under a confocal laser scanning microscope (Leica TCS SP5; Leica Microsystems, Mannheim, Germany).
For the transactivation activity assay, the LaMYC7 sequence was recombined into the pGBKT7 vector.The recombinant (pGBKT7-LaMYC7), positive control (pGBKT7-p53), and negative control (pGBKT7) vectors were transformed and expressed in AH109 yeast cells.

Sequence analysis of LaMYC7
Arabidopsis bHLH protein (AtbHLH) sequences were acquired from TAIR database (http://www.arabidopsis.org).Two specific MYC domains PF14215 and PF00010 were used to query the L. angustifolia genome database (PRJNA642976).Based on the AtbHLH and LaMYC protein sequences, MEGA 7.0 software was used to con-struct a phylogenetic tree using the neighbor-joining method with 1000 replicates.Characteristics of the LaMYC7 protein sequence were determined by the Compute pI/Mw tool (ExPASy; https:// web.expasy.org/compute_pi/).For the functional prediction analysis of cis-regulating elements, the promoter sequence (2000-bp upstream of the translation initiation site) of the LaMYC7 gene was submitted to PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

qRT-PCR analysis
qRT-PCR was performed on the Stratagene Mx3000P system (Agilent Technologies, Palo Alto, CA, USA).The PCR procedure and data analysis were completed as previously described [52].

Plant transformation
Using the leaf disk method for Nicotiana [53] or the f loral dip method for Arabidopsis [54], bacterial colonies bearing the 35S::LaMYC7-GFP vector were selected and transformed.Plants infiltrated with the empty vector were used as control.Transgenic
LaTPS76 isolated from sepal cDNA by PCR was recombined into the pCAMBIA2300 vector.LaTPS76-overexpressing transgenic Arabidopsis plants were obtained by A. tumefaciens-mediated transformation.Terpenoid levels were analyzed in the T3 generation.Sequences and primers of LaTPS26 and LaTPS76 are shown in Tables S3 and S4.

Measurement of volatile terpenoid content
Volatile compounds emitted by Arabidopsis and Nicotiana were collected by SPME as previously described [39].Samples were injected in the splitless mode.Products were identified based on retention times, electron ionization mass spectra from the NIST Mass Spectral Library (NIST-14.0),and information from the literature [34,57].

Dual-luciferase (dual-LUC) assay in tobacco leaves
For the dual-LUC assays, the LaTPS76 promoter was inserted into the pGreenII 0800-LUC vector as a reporter.The effector and reporter were transformed into A. tumefaciens strain GV3101 with the helper plasmid pSoup-19.Agrobacterium harboring the reporter vector was then co-infiltrated into N. benthamiana leaves with Agrobacterium carrying either the LaMYC7-overexpressing vector (pCAMBIA2300-LaMYC7) or the control vector (pCAM-BIA2300) in a 1:1 ratio.After incubation at 22 • C for 36 hours, the agro-infiltrated leaves were collected, and the FLuc activity was quantitatively analyzed using a dual-LUC assay kit (Yeasen Biotechnology, Shanghai).The analysis was performed using the GloMax 20/20 luminometer (E5311; Promega) according to the manufacturer's instructions.At least five measurements were performed for each assay.

Measurement of TAC and endogenous hormone contents
Twelve plants from each sample were selected to assess plant height and TAC.TAC in 500 mg of Nicotiana f lowers was measured as previously described [31].The ZR, IAA, JA, GA 3 , and ABA levels were measured in Nicotiana leaves using ELISA.Hormones were isolated, purified, and quantified by ELISA as described by He [59] and Yang et al. [60].

Assessment of pathogen and drought tolerance in transgenic plants
The stress resistance abilities of WT, empty vector (2300), and transgenic (#2 and #9) plants were investigated.To simulate drought stress, four-week-old potted Nicotiana plants were grown without water for 45 days in a greenhouse.Plants were given sufficient water to rehydrate, and they were observed and photographed the following day.For pathogen infection treatment, 8-week-old potted Nicotiana plants were inoculated with Pst DC3000.Pst DC3000 solution for inoculation was prepared by the method of Chen et al. [61].The WT, 2300, #2, and #9 plants were sprayed with Pst DC3000 solution.Five days after Pst DC3000 inoculation, six leaves from a single plant were harvested and rinsed twice with sterile water.The leaf-infected regions were excised using a hole punch, and the disks were homogenized in Luria Broth containing rifampicin and then cultured on solid medium for 2 days at 28 • C.

Figure 1 .
Figure 1.LaMYC7 characteristics.(A) Transcriptional alterations of LaMYC7 from sepal or leaf with methyl jasmonate treatment (CKS, JAS, CKL, and JAL: CK, control; JA, methyl jasmonate treatment; S, sepal; L, leaf).(B) LaMYC7 expression levels in L. angustifolia tissues (LAR, root; LAS, stem; LAL, leaf; LAF, f lower; LAGT, glandular trichome).(C) Transcription abundance of LaMYC7 at different stages (FB0, FB1, FB2, F3, F4, and F5; 'F' means f lower, 'FB' means f lower bud and '1-5' five degrees of maturity).(D) Evolutionary tree analysis of LaMYC7 and AtMYC TFs.The method of neighbor-joining was used on MEGA7.0 to build the evolutionary tree, and 1000 replications of the bootstrap method were used to calculate the bootstrap values.(E) The NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used to analyzed conserved domains.The numbers displayed are the average of at least three replicates (mean ± SD).The top of each bar represents standard errors, and bars annotated with different letters were significantly different according to Fisher's LSD test (P < 0.05) after ANOVA.

Figure 2 .
Figure 2. Analysis of LaMYC7 protein.(A)Subcellular localization used tobacco leaves.35S::GPP was empty vector.35S::LaMYC7-GFP was full length CDS of LaMYC7 recombined into the pCAMBIA2300 vector.The transformed tobacco leaves were then stained with 10 g/ml DAPI in order to be visualized.(B) Yeast AH109 cells with the positive control pGBKT7-p53, the recombined pGBKT7-LaMYC7, and the negative control pGBKT7 are shown in the top, middle, and bottom panels, respectively.

Figure 3 .
Figure 3. Analysis of the LaMYC7-overexpressing in tobacco.Wild-type (WT) plants transformed with the empty vector pCAMBIA2300 (2300) and LaMYC7-overexpressing plants with 35S::LaMYC7-GFP (#2, #9).(A-D) GC trace of caryophyllene and linalool.The peak area was indicated by the number on the peak.(E) Linalool content.(F) Caryophyllene content.(G-M) Relative expression levels of NtHMGRL, NtFPPS, NtDXS, NtDXR, NtGPPS, NtTPS76 and NtTPS7.By comparing the products to substances in the NIST14 collection and reference standards, the compounds were identified.The numbers displayed are the average of at least three replicates (mean ± SD).Following an ANOVA, Fisher's LSD test revealed that bars labeled with various letters were significantly different (P < 0.05), as seen by the vertical lines at the top of each bar indicating standard errors.

Figure 4 .
Figure 4. Analysis of the potential biological role of LaMYC7 for P. syringae.A, P. syringae infection of WT, 2300, and LaMYC7-overexpressing transgenic lines for 5 d for phenotype analysis.B, C, Bacterial population at 5 d in WT, 2300 and LaMYC7-overexpressing transgenic lines (#2, #9).D, E, Antibacterial activity of linalool and caryophyllene against Pst DC3000.LB, Empty lysogeny broth; Pst DC3000, 150 μl Pst DC3000 were dissolved in lysogeny broth medium; Lin + Pst DC3000, 150 μl Pst DC3000 were dissolved in lysogeny broth medium containing 18 μl•ml −1 linalool.Car + Pst DC3000, 150 μl Pst DC3000 were dissolved in lysogeny broth medium containing 18 μl•ml −1 caryophyllene.The numbers displayed are the average of at least three replicates (mean ± SD).Following an ANOVA, Fisher's LSD test revealed that bars labeled with various letters were substantially different (P < 0.05), as seen by the vertical lines at the top of each bar indicating standard errors.

Figure 5 .
Figure 5. Sequence analysis and subcellular localization of LaTPS26 and LaTPS76.A, Multiple alignment of TPSs.B, Subcellular localization of LaTPS26 and LaTPS76 in Nicotiana leaves.

Figure 8 .
Figure 8. Analysis of the promoter and expression level of LaMYC7 and the changes of linalool and caryophyllene contents under various stresses.A, Analysis of the LaMYC7 promoter sequence.The PlantCARE database was utilized to find potential cis-acting regulatory elements.B, Relative expression of LaMYC7 was measured using qPCR.C, Linalool content under various stresses.D, Caryophyllene content under various stresses.The number displayed are the average of at least three replicates (mean ± SD).Following an ANOVA, t test revealed that bars labeled with * (p < 0.05), * * (p < 0.01), * * * (p < 0.001) were significantly different, as seen by the vertical lines at the top of each bar indicating standard errors.